6533b870fe1ef96bd12cf079

RESEARCH PRODUCT

Activation and Inactivation of Tetanus Toxin in Chromaffin Cells

subject

description

Tetanus toxin is produced by Clostridium tetani as a single chain, almost non-toxic, protein with a molecular weight of approximately 150.000 representing 1315 amino acids. Bacterial proteases cleave the molecule between positions A 457 and S 458 (extracellular activation), yielding a heavy chain (MW 100.000) and a light chain (MW 50.000) tetanus toxin (HC-TeTx, LC-TeTx). Both chains remain connected to each other by a disulphur bond between positions C 439 and C 467 (Dichain-TeTx)1. The cleavage or nicking dramatically increases the biological activity2. HC-TeTx is involved in binding DC-TeTx to gangliosides lodged in the plasma membrane, which is a prerequisite for incorporation into the cytosol3. Whether DC-TeTx diffuses into the cells through pores formed by H-CTetx4 or is taken up by receptor-mediated endocytosis is not yet clear. Within the cells, the disulphur bond between the two chains is reduced to separate LC-TeTx from DC-TeTx (intracellular activation). Toxicity is carried exclusively by the reduced form of LC-Tetx5. The intracellular target of the toxin is present in several types of neuronal and endocrinal cells, such as cholinergic, noradrenergic, glycinergic, GABAergic, and other neurons6 and also chromaffin cells7. Therefore they all respond to the toxin. However, since chromaffin cells lack gangliosides in their plasma membrane8 they cannot take up the toxin unless special manipulations are performed. Nevertheless, chromaffin cells have firmly been established as useful tools for studying the interference of TeTx with exocytosis, because they represent a homogenous population and offer an excellent access to structures controling hormonal release. There are several ways to introduce TeTx into their cytosol. It can diffuse through plasma membrane pores induced by cytolysins5, and it can be injected7, or taken up by exogenous gangliosides incorporated into the plasma membrane9. Cytolysinepermeabilised chromaffin cells, in contrast to ganglioside-enriched or micropipette-porated cells, respond only to LC-TeTx or to chemically reduced DC-Tetx5, indicating that, during their short survival time, these cells are not able to cleave the sulphur bonds.